Legionella pneumophila is a Gram negative bacterium that replicates inside fresh water amoebae in the environment but can also cause Legionnaires'disease in humans by replicating inside alveolar macrophages. L. pneumophila survives and replicates inside normally bactericidal phagocytic cells by altering their endocytic pathway, thereby inhibiting rapid phagosome-lysosome fusion. L. pneumophila is able to perturb normal host cell function by injecting a large number of virulence factors into the host via a type IV secretion system (T4SS). The L. pneumophila T4SS is encoded by twenty six dot or icm genes, which encode a large macromolecular complex in the bacterial cell envelope. Due to the complexity of this system, the molecular mechanism of how L. pneumophila exports substrates has remained undetermined. However, a recent advance in the field of bacterial secretion has provided a new and powerful technique, TrIP (translocation immunoprecipitation), that allows a detailed understanding of substrate secretion by T4SSs. Using TrIP, not only can specific Dot/lcm proteins be shown to interact with a translocated substrate, but the timing of their interactions in relation to each other can be ascertained. Based on this advance, our overall goal is to understand how the Dot/lcm secretion system functions. This research will relate to human health in two ways. First, establishing the mechanism used by L. pneumophila to survive and replicate inside macrophages will provide additional insight into how it causes disease and may reveal novel targets to be used for drug therapy. Second, since specialized secretion systems are commonly used by a variety of bacterial pathogens, knowledge gained about the L. pneumophila secretion apparatus is likely to be applicable to understanding the molecular mechanisms of virulence used by other pathogens, and could serve as the basis to prevent or treat a number of different diseases.